Project Summary/Abstract This application seeks to understand the mechanism of breast cancer?s relative resistance to immunotherapy, including immune checkpoint inhibitors, and to identify potential targets to overcome this resistance. Immune escape is a hallmark of disease progression and metastasis and a major impediment to designing efficacious anticancer therapies. Breast cancer is considered less immunogenic, and thus relatively resistant to immunotherapy such as immune checkpoint inhibitors (ICI), although there is evidence of only a modest response to ICIs in patients with triple negative breast cancer (TNBC) compared to other breast cancer subtypes. The exact mechanism for this relative resistance of breast cancer to ICIs remains unclear. However it is increasingly clear that recruiting inhibitory immune cells such as regulatory T cells (Treg) to the tumor microenvironment is an effective way tumors employ to suppress effective anti-tumor immunity and promote immune escape. In this proposal, we describe the application of a novel computational approach to identify and validate the underlying tumor cell-intrinsic molecular determinants of intratumoral Treg stability and immune escape. Our objective is to identify novel targets in breast cancer to prevent immune escape and to enhance breast cancer?s response to ICIs. To that end we have discovered a novel subnetwork regulated by Snail1, a master epithelial-to-mesenchymal transition (EMT) initiating factor, which is predicted to confer an immune surveillance program by suppressing the intratumoral Treg population. The central hypothesis of this proposal is that Snail1 negatively regulates Pglyrp3, a protein known for its Treg recruitment functions, therefore limiting the Treg population, enhancing anti-tumor immunity and response to ICIs. In Aim 1, we will determine the impact of the Snail1-Pglyrp3 interaction on intratumoral Treg stability using breast cancer models. Specifically, we will manipulate levels of Pglyrp3 in Snail1-positive or -negative backgrounds and determine how the intratumoral Treg population is impacted and whether additional factors are required for the Snail1-Pglyrp3 interaction to be functional. In Aim 2, we will determine whether targeting the Snail1-Pglyrp3 interaction is sufficient at enhancing response to immune checkpoint inhibitors and improving survival. This work is significant because it will demonstrate a novel network involved in switching from an immune-active to immune-suppressed tumor microenvironment that shapes tumor evolution. This work is innovative in that it will provide novel targets that can be pursued to prevent breast tumors from immune evasion and to sensitize them to ICIs.